Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
exposure considerations
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
data waiving: supporting information
Reference
Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - oral (%):
10
Absorption rate - dermal (%):
10
Absorption rate - inhalation (%):
100

Study 1. A study was conducted to determine the basic toxicokinetics of the test substance according to EPA OPP 85-1, in compliance with GLP. Sprague-Dawley rats (10 animals per sex per group) were treated with radiolabelled test substance. The study was conducted in four experiments: a single low dose (10 mg/kg); a single high dose (50 mg/kg); a 14-d repeated dietary exposure with non-radiolabelled test substance (100 ppm) and single low dose of radiolabelled (14C) test substance (10 mg/kg); and a single intravenous dose (10 mg/kg). Following the single doses or the last dietary dose, urine and faeces were collected for 7 d. Tissues, urine and faeces were collected and analysed for radioactivity and faeces were analysed by TLC, HPLC and MS for metabolites and parent compound. Following oral administration, radiolabelled test substance was rapidly absorbed, although in very limited amounts, consistent with its highly ionic nature. Residual 14C in tissues was negligible after administration by gavage both after single and repeated dosing, indicating low potential for bioaccumulation. After i.v. administration, a higher amount of radioactivity (30−35%) was found as residue in the tissues. Approximately 6−8% of orally administered test substance was excreted in the urine whereas 87−98% was found in the faeces. Since no data on bile duct-cannulated rats are available, it was not possible to conclude if this radioactivity accounted exclusively for unabsorbed test substance or not. However, the i.v. experiment showed that 20−30% was excreted in urine and 44-55% in faeces, suggesting that both the kidney and liver were capable of excreting test substance once absorbed and that absorption was higher than the % found in the urine after oral administration. Less than 50% of the orally administered test substance was metabolised to side-chain oxidation products. In view of the limited absorption of the test substance, the four major metabolites identified may be at least have been partially formed in the gut of rats, apparently by microflora. No significant difference in metabolism between male and female rats or amongst dosing regimens was observed. Repeated dosing did not alter uptake, distribution or metabolism of the test substance (Selim, 1987).

Study 2. A study was conducted to determine and predict the most likely site of metabolism and the metabolites of the test substance using the ECETOC recommended Metaprint2D-React tool (ALL metabolite model: 2010.2). The modelling was conducted using SMILES, as the input parameters. The most frequently reported site of Phase I metabolism occurring at the terminal two carbon atoms of the longer alkyl chain were oxidation at (keto hydroxy, single or double bond, ketone and di ketone group), alkylation, methylation, acylation, demethylation and dealkylation. Also, the reactions occurring at the para-position of the benzene ring included hydroxylation, methoxylation and glutathionation (MetaPrint2D-React, 2013).

Study 3. A study was conducted to determine the basic toxicokinetics of the test substance according to OECD Guideline 417, in compliance with GLP. This toxicokinetic study was conducted using radiolabelled test substance. Rats were treated with single and repeated oral doses (50 or 200 mg/kg bw) as well as a single dermal dose of 1.5 or 15 mg/kg bw. Following single and/or repeated oral doses, the plasma, blood and organ radioactivity levels were essentially non-quantifiable, indicating a low oral bioavailability. The actual fraction of the oral dose absorbed was around 8% (urine and bile fractions). This was eliminated rapidly, essentially within a 48 to 72 h period. The majority of the oral dose was excreted in the faeces. At the high oral dose level only, quantifiable levels of radioactivity (2,386 to 23,442 ηg equivalent/g) were found in some central organs at 8 h post-dosing; otherwise, the vast majority of the dose was confined to the intestines and levels decreased over time. Only about 4% of the oral dose was eliminated in the bile in a 24 h period, of which about 30% during the first 3 h. Following a single dermal application, the plasma and blood radioactivity levels were non-quantifiable at nearly all time-points. For the 1.5 mg/kg bw group, around 2 and 43% of the dose was eliminated in the urine and faeces, respectively, mostly within a 48 h period, suggesting that the dermal dose was highly absorbed via the skin. However, this apparent high absorption via the skin may have been due to the animal licking the test site. This is also supported by the finding that, after oral dosing, only about 4% was excreted via bile back to the intestine and 4% excreted via urine. If similar routes of excretion are expected for dermally absorbed doses, it would not be possible to find levels of 50% of applied doses in intestine with only 2% excreted via urine. This indicates that about 50% of the dermally applied dose was taken up orally after all. According to the same oral kinetics, this leads to the 2% excretion in urine as indeed was observed. At 24 h post-dosing, most of the radioactivity was in the “stripped” skin (dermis/epidermis) application site (15.02/8.74% [male/female] and 33.8/24.2% of the dose for the high and low dose groups respectively) and intestines for both dose levels (5.76/8.32% and 5.61/7.79% of the dose for the high and low dose groups respectively), though some radioactivity was in the skin adjacent to the application site and minor traces were in the eyes (both most likely from cross-contamination due to grooming). At 168 h, levels in the application site of the individual animals of the low dose were 5.19 to 9.21% of the radioactive dose, suggesting the skin acted as a drug reservoir. In the stratum corneum of the application site, the levels of radioactivity were of similar magnitude in the different layers at each time-point. For all tissues/organs, the radioactivity levels decreased over time (Appelqvist, 2006).

Study 4.Studies conducted in rat and Beagle dogs compared the application of test substance in both milk and water as vehicle. Rats received 50 and 100 mg/kg bw/d for 12 weeks, and dogs 12.5, 25 and 50 mg/kg bw/d for 52 weeks. Depression in weight gain was observed in rat receiving 100 mg/kg bw/d in water, but not in milk. Mortality occurred in dogs at 25 and 50 mg/kg bw/d in water, but not in milk. The 12.5 mg/kg bw/d dose level was well tolerated (Cutler and Drobeck, 1970).

Study 5. A study was conducted to evaluate the distribution of the test substance inside the body of rabbits, cats and dogs. A single high dose (approximately 1 mL of 15% solution of the test substance in water/kg bw of animals, which is equal to about 10 times the lethal dose) was administered by oral, rectal and intramuscular route. Within a few minutes, the animals died and the test substance concentrations were measured locally at the sites of dosing, in blood, liver and kidneys. It was concluded that only a small fraction is absorbed and distributed in the body. Less than 1.5% of the applied dose was found in the organ investigated (sites of dosing, blood, liver and kidneys), of which the major part was found in the liver (Bogs, 1971).

Dermal absorption

Study 6.A study was conducted to evaluate the dermal penetration of the test substance in normal excised human skin. From un-buffered aqueous solutions of the test substance ranging in concentration from 0.005 (1.7 ppm) to 0.1 M (34 ppm), no measurable amount penetrated into the dermis of excised human skin within periods of 1 to 3 d at temperatures between 23 and 35°C. Lowering the pH of the contact solution up to pH 1.3 had no influence. However, at pH 10.5 to 12, the test substance could be recovered from the skin. At these levels, electrical conductivity indicated damage of the cutaneous barrier. Similarly, pre-treatment of skin at that pH caused damage of the skin and resulted in penetration of the test substance upon subsequent contact to test solutions. Also, damaging the skin by repeated stripping of the stratum corneum with pressure-sensitive tape resulted in penetration into the skin when stripped more than 10 times (Blank, 1964).

Study 7.A study was conducted with single or multiple drops of radiolabelled test substance on the cornea of rabbits to determine corneal penetration. The test substance was found in the palpebral and bulbar conjunctiva, corneal epithelium, stroma and endothelium. Single-drop administration resulted in high tissue levels in the anterior ocular tissues that were retained for up to 120 h. Multiple-drop administration led to accumulation in the epithelium to a greater degree than any other tissue. However, at no time did the test substance appear in the aqueous humour or any other tissue besides cornea and exposed conjunctivae (Green and Chapman, 1986).

Study 8. A study was conducted to determine the dermal absorption of the test substance according to OECD Guideline 428, in compliance with GLP. In thein vitrostudy, split-thickness human skin membranes were mounted into flow-through diffusion cells. Receptor fluid was pumped underneath the skin at a flow rate of 1.5 mL/h. The skin surface temperature was maintained at approximately 32°C. A barrier integrity test using tritiated water was performed and any skin sample exhibiting a permeability coefficient (kp) greater than 2.5 x 10-3cm/h was excluded from subsequent absorption measurements. Two test preparations containing (14C)-radiolabelled test substance (0.03 and 0.3%), were applied at an application rate of 10 mg/cm2. Absorption was assessed by collecting receptor fluid in hourly intervals from 0-6 h post dose and then in 2-hourly intervals from 6-24 h post dose. At 24 h post dose, the exposure was terminated by washing and drying the skin. The stratum corneum was then removed from the skin by 20 successive tape strips. All samples were analysed by liquid scintillation counting. Under the study conditions, following topical application of (14C)-radiolabelled test substance in low (0.03%, w/w) and high (0.3%, w/w) concentration test preparations to human skinin vitro, the mean absorbed dose and mean dermal deliveries were 0.05% (0.01 ηg equiv. /cm2) and 2.22% (0.07 ηg equivalent/cm2) of the applied dose for the low concentration test preparation, respectively, and 0.03% (0.01 ηg equivalent /cm2) and 2.16% (0.67 ηg equivalent/cm2) of the applied dose for the high concentration test preparation, respectively. The stratum corneum acted as a barrier to absorption, with the mean total unabsorbed doses (recovered in skin wash, tissue swabs, pipette tips, cell wash, stratum corneum and unexposed skin) of 96.80 and 94.68% of the applied dose for the low and high concentration test preparations, respectively. The maximum fluxes for the low and high doses were 0.12 ηg equivalent /cm2/h and 0.74 ηg equivalent /cm2/h, respectively, at 2 h (Roper, 2006).

Study 9.A study was conducted to determine the dermal absorption of the test substance a method comparable to OECD Guideline 427, in compliance with GLP. The dermal absorption and excretion study was conducted in rats following application of 0.4 mL of a 0.77% w/w solution of the test substance over approximately 20 cm2 of shaved skin, under a gauze patch for 72 h. After a single topical application of radio-labelled test substance, the total amount of radioactive substance was 16% in males (urine 0.8%, faeces about 9.9% and carcass about 5.3%) and 14% in females (urine about 0.7%, faeces about 6.1% and carcass about 7.0%). This was equivalent to a total mass of 24 µg equivalents (males) and 21 µg equivalents (females) absorbed per cm2(after a dose of approximately 3 mg). Most of the radio-labelled test substance (62.6%, males; 63.2%, females) was found in both the treated (48.0%, males; 45.1%, females) and the untreated (14.6%, males; 18.1%, females) skin after 72 h. The radioactive substance in the untreated skin may have been due to surface migration of the applied material from the perimeter of the treated area. The overall recoveries of radioactivity were acceptable for the experimental objectives of quantifying the absorption of radio-labelled test substance after a single dermal application. Under the study conditions, the findings indicate that the dermal absorption of the test substance is limited and most of the absorbed test substance is excreted in the faeces (Hallifax, 1991).

Reason / purpose:
data waiving: supporting information
Reference

The test substance is considered to be corrosive to skin and eyes.

Endpoint conclusion:
adverse effect observed (corrosive)
Endpoint conclusion:
adverse effect observed (irritating)

Skin

Study 1. A study was conducted to determine the skin irritation / corrosion potential of the test substance according to the method 'Transport of dangerous goods, special recommendations relating to Class 8, United Nations handbook, 1977'. In this experiment, 0.5 mL of a 50% solution of test substance was applied under occlusive dressing to the skin of 1 rabbit for 3, 30, 60 min and 4 h. The skin was washed with water upon removal of the dressing. Observations were recorded at 24, 48 and 72 h. A confirmatory study was performed with 3 min or 1 h applications in 3 rabbits each. In the main study, no dermal reactions were observed at any of the 6 sites after 3 min application. Moderate erythema with slight oedema at 4 sites and areas of skin necrosis at the other 2 sites were observed following 1 h application (Primary irritation index PII: 3 min: 0; 60 min: 4.5). Under the conditions of the study, the test substance solution was considered to be corrosive to rabbit skin (Liggit, 1982).

Study 2. A study was conducted to determine the skin irritation / corrosion potential of the test substance according to the method 'U. S. Federal Register, Vol. 41, No. 188, P. 42572’. The experiment was performed in rabbits. Six animals were treated with 0.5 mL of the test substance for 4 h under occlusive conditions. As the test proved positive for corrosion after 4 h, it was repeated in a different group of animals for an exposure period of 60 min and then for an exposure period of 3 min. Under the test conditions, the substance was corrosive to rabbit skin (Sugar, 1981).

Study 3. A study was conducted to determine the skin irritation / corrosion potential of the test substance according to US EPA OPPTS 870.2500. The experiment was performed in rabbits. The undiluted test substance was applied on intact and abraded skin sites using occlusive patches for an exposure period of 24 h. The skin was then observed for erythema and edema formation and the scoring was done according to the Draize, Woodland and Calvery scoring system at 24 and 72 h from the onset of exposure. Severe erythema and edema were observed in all the test animals at both the abraded and intact sites. The mean Primary Irritation Index (PII) of the test substance was calculated to be 6.29. The mean values of erythema and edema were 3.33 (intact skin site), 3.5 (abraded skin site), 2.66 (intact skin site) and 3 (abraded skin site). The test substance was irritating to rabbit skin (Wallace, 1975).

Study 4. A study was conducted to determine the skin irritation / corrosion potential of the test substance according to OECD Guideline 404. Skin of albino rabbit was used in this experiment. A single 24 h, occluded application of the test substance (undiluted) to the intact and abraded skin of six rabbits produced necrosis with blanching extending beyond the entire site and severe oedema. Forty-eight hours after patch removal, each site and beyond was coriaceous and slight oedema was noted. No further observations were made. Corrosive effects were noted in all 6 rabbits. Under the study conditions, the test substance was corrosive to rabbit skin (Anspach, 1976).

Study 5. A study was conducted to determine the skin irritation / corrosion of the test substance according to OECD Guideline 404. The experiment was performed to assess in albino rabbits (strain not specified). Under the study conditions, a 0.1% aqueous solution of the test substance was not irritating to rabbit skin (following 24 h exposure under occluded conditions) (Hixson, 1968).

Study 6. A study was conducted to determine the skin irritation / corrosion potential of the test substance according to a method similar to OECD Guideline 404, in compliance with GLP. The experiment was performed with a 25.5% active test substance. The objective of this study was to define a clear "non-irritant" level of administration and the maximum tolerated test concentration (irritancy, but not severe irritancy, corrosivity, toxicity or mortality) following a single topical administration to Wistar rats, and thereby select dosages for an absorption, distribution and excretion study in the same strain of rat. Four groups of five male and five female Wistar rats were treated with 240 mm3 aliquot of 2.55, 1.28, 0.77 and 0.26% w/v test substance. The test site was protected by an Elizabethan collar for a period of 72 h following administration. Under the conditions of this study, the 'threshold of irritancy' of the test substance in distilled water was between 0.26 and 0.77% w/v, and the 'maximum tolerated' concentration slightly in excess of 2.55% w/v. The lowest tested concentration of 0.26% w/v was 'non-irritant' to rabbit skin (Cummins, 1991).

Study 7. A study were conducted to determine the skin irritation / corrosion potential of the test substance. When applying 0.5 mL of test substance on rabbit skin, concentrations of 1% or greater induced skin reactions, while 0.1% was not irritating. Exposure for 24 h to 0.1% aqueous test solution was not irritating to guinea pig skin, while the same exposure regime of 0.3% concentration caused irritation in rabbits. Also, 0.5 mL of a 0.5% aqueous test solution on the skin of nine rabbits for 24 h under occlusive patch, resulted to barely perceptible erythema in one animal and no reactions in the others. In another study, 0.3% test solution was tested according to the same protocol. Skin irritation was not observed in any of the nine rabbits. A 0.1% aqueous solution of test substance was applied to the skins of rabbits under plastic wrap for 5 d. At the end of the period, necrosis and varying degrees of erythema with diffuse areas of eschar and bleeding were noted. Data from published literature suggested that test substance was corrosive to rabbit skin (BIBRA, 1989; CIR, 1989).

Eye

Study 1. A study was conducted to determine the eye irritation / corrosion potential of the test substance (purity not mentioned) according to a method similar to OECD Guideline 405. The experiment was performed in albino rabbits. A single application of the undiluted (purity not mentioned) test substance to the non-irrigated eye of 6 rabbits produced severe corneal opacity, conjunctivitis and blanching of the conjunctival membranes. Iritis could not be scored due to the severe corneal opacity. Crystallization and fissuring were noted in one or two animals. The animals were observed for 3 days after application. Under the study conditions, the undiluted test substance produced serious eye damage to rabbits and needs classification as an eye irritant (Anspach, 1976).

Study 2. A study was conducted to determine the eye irritation / corrosion potential of the test substance according to a method similar to OECD Guideline 405. The experiment was performed in albino rabbits. A single application of the test substance as a 0.1% aqueous solution to the non-irrigated eye of nine rabbits produced moderate effects on the conjunctivae 1 h after application, but these had disappeared within 1 day. The animals were observed for 7 days after application. Under the study conditions, the test substance as a 0.1% aqueous solution was not irritating to the rabbit eye (Hixson, 1968).

Study 3. A study was conducted to determine the eye irritation / corrosion potential of the test substance according to a method similar to OECD Guideline 405. The experiment was performed in New Zealand rabbits. A single application of the undiluted test substance (purity not mentioned) to the eye of 6 rabbits produced severe corneal opacity, iritis, and moderate erythema and chemosis. There was a delayed occurrence of the effects, therefore, the mean values on Days 5 -7 were chosen for the evaluation. Under the study conditions, the test substance produced serious eye damage to rabbits (Sterner, 1981).

Study 4. A study was conducted to determine the eye irritation / corrosion potential of the test substance according to a method similar to OECD Guideline 405. The experiment was performed in rabbits. All six test rabbits received 0.1 mL of the undiluted test substance in one eye. The other eye remained untreated. Eyes were not washed throughout the study. After 24, 48 and 72 h, eyes were evaluated for ocular lesions according to the Draize scale. Under the conditions of the study, the test substance produced severe and irreversible damage in rabbit eyes (Wallace, 1975).

Study 5. Due to the direct corrosive effect, there is danger of irreversible damage to the eyes upon exposure to the undiluted solution. Testing for acute eye irritation is, due to its corrosive effects, not ethical. It has been established (CIR, 1989) that concentrations above 1% and higher caused severe damage of the rabbit eye upon twice daily instillation for 7d (CIR, 1989).

Skin

In an in vivo skin irritation study, there was evidence of skin necrosis when 50% active test substance was applied under occlusion to intact rabbit skin for a period of 60 minutes. Hence, the data justifies classification as Skin Corr. 1B; H314: causes severe skin burns and eye according to CLP (EC 1272/2008) criteria.

Eye

Undiluted test substance (0.1 mL) produced severe and irreversible damage in the eyes. Also, the test substance is skin corrosive. The data justifies classification as Eye Damage 1-H318: Causes serious eye damage according to CLP (EC 1272/2008) criteria.

Data source

Materials and methods

Results and discussion

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion